Biotechnology has emerged as an essential tool in efforts to meet the challenge of increasing global demand for food production. Conventional approaches to improving agricultural productivity, e.g. enhanced yield or engineered pest resistance, rely on either mutation breeding or introduction of novel genes into the genomes of crop species by transformation. Both processes are inherently nonspecific and relatively inefficient. For example, conventional plant transformation methods deliver exogenous DNA that integrates into the genome at random locations. Thus, in order to identify and isolate transgenic lines with desirable attributes, it is necessary to generate thousands of unique random-integration events and subsequently screen for the desired event. As a result, conventional plant trait engineering is a laborious, time-consuming, and unpredictable undertaking. Furthermore the random nature of these integrations makes it difficult to predict whether pleiotropic effects due to unintended genome disruption have occurred. As a result, the generation, isolation and characterization of plant lines with engineered transgenes or traits has been an extremely labor and cost-intensive process with a low probability of success.
Targeted gene modification overcomes the logistical challenges of conventional practices in plant systems, and as such has been a long-standing but elusive goal in both basic plant biology research and agricultural biotechnology. However, with the exception of “gene targeting” via positive-negative drug selection in rice or the use of pre-engineered restriction sites, targeted genome modification in all plant species, both model and crop, has until recently proven very difficult. Terada et al. (2002) Nat Biotechnol 20(10):1030; Terada et al. (2007) Plant Physiol 144(2):846; D'Halluin et al. (2008) Plant Biotechnology J. 6(1):93.
Recently, methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted mutations (e.g., deletions, substitutions and/or insertions) of cellular DNA sequences, and facilitate targeted recombination and integration at a predetermined chromosomal locus. See, for example, Urnov et al. (2010) Nature 435(7042):646-51; United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20090263900; 20090117617; 20100047805; 20110207221; 20110301073; 2011089775 and International Publication WO 2007/014275, the disclosures of which are incorporated by reference in their entireties for all purposes. Cleavage can occur through the use of specific nucleases such as engineered zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs), homing endonucleases, or using the CRISPR/Cas system with an engineered crRNA/tracr RNA (‘single guide RNA’) to guide specific cleavage. U.S. Patent Publication No. 20080182332 describes the use of non-canonical zinc finger nucleases (ZFNs) for targeted modification of plant genomes; U.S. Patent Publication No. 20090205083 describes ZFN-mediated targeted modification of a plant EPSPS locus; U.S. Patent Publication No. 20100199389 describes targeted modification of a plant Zp15 locus and U.S. Patent Publication No. 20110167521 describes targeted modification of plant genes involved in fatty acid biosynthesis. In addition, Moehle et al. (2007) Proc. Natl. Acad, Sci. USA 104(9):3055-3060 describes using designed ZFNs for targeted gene addition at a specified locus.
Carbon assimilation is central to the metabolic functioning of all living organisms. The ability to synthesize ATP and utilize its energy for homeostasis, growth and reproduction is conserved across kingdoms and impacts a majority of known biological processes. A fundamental component of ATP synthesis in eukaryotes is the tricarboxylic acid (TCA) cycle, also known as the citric acid or Krebs cycle, which moves electrons from organic acids to the oxidized redox cofactors NAD+ and FAD, forming NADH, FADH2 and carbon dioxide. The TCA cycle takes place within mitochondria; in plants, intermediates produced during its reactions serve as substrates for numerous biosynthetic pathways; primary inputs for the production of aspartate, glutamate, nucleic acids, porphyrins and fatty acids originate from the TCA cycle. In addition, TCA cycle intermediates play a key role in the energetic processes of photorespiration and photosynthesis. Therefore, the TCA cycle is thought to act as a link between chloroplastic, mitochondrial and cytosolic redox functions.
Malate is one of the intermediates of the TCA cycle and acts as a substrate for both malic enzyme, which generates pyruvate, and malate dehydrogenase (MDH). MDH catalyzes the reversible reduction of oxaloacetate (OAA) to malate via NADH and is involved in the malate/aspartate shuttle. Most plants contain multiple isoforms of MDH, including mitochondrial and cytosolic enzymes, which are encoded by nuclear genes. The plant mitochondrial MDH (mMDH) participates in 3 types of reactions: conversion of malate to OAA, reduction of OAA to malate, and C4-pathway reduction of OAA. In maize (a C4 grass), there are 5 distinct MDH loci on 5 independent chromosomes, 2 of which encode cytosolic isoforms while the other 3 encode mitochondrial enzymes. Using classical mutant analyses, it was demonstrated that complete loss of function of the 2 cytosolic forms of MDH had no deleterious effects on plant growth and reproduction—the cytosolic function appeared to be dispensable. In contrast, complete loss of the 3 mitochondrial enzymes resulted in lethality—the plants needed at least one functional allele in order to be viable (Goodman et al. (1981) Proc. Nat. Acad. Sci. USA 78:1783-1785). Similarly, observations of naturally occurring spontaneous null alleles of mitochondrial MDH-1 (Mdh1-n) in soybean showed that there was no obvious plant phenotype as long as the mitochondrial Mdh2 gene remained stable (hnsande et al. (2001) J Heredity 92:333-338).
Despite its fundamental role in plant metabolism, the functions of malate in the TCA cycle are still not completely understood. MDH-mutant plants exhibit slower growth rates and altered photorespiratory characteristics. See, e.g., Tomaz et al. (2010) Plant Physiol. 154(3):1143-1157. Anti-sense and RNAi studies in whole plants or fruit have shown contradictory results, including plants with increased dry (not fresh) fruit weights as well as plants having higher ascorbate levels in their leaves than wild-type controls but, when grown under short-day light conditions (which favor photorespiration), the plants displayed a dwarf phenotype and had reduced biomass in leaves, stems and roots. Nunes-Nesi et al. (2005) Plant Physiol. 137: 611-622); Nunes-Nesi et al. (2007) Physiol. Plant. 129:45-56); Nunes-Nesi (2008) J. Exp. Bot. 59:1675-1684; Finkmeier and Sweetlove (2009) F1000 Biology Reports 1:47; doi:10.3410/B1-47. Furthermore, mMDH anti-sense lines with reduced mMDH expression exhibited reduced activity (39% of wildtype) of this enzyme resulted in decreased root area and stunted root growth. Van der Merwe et al. (2009) Plant Physiol. 149:653-669); Van Der Merwe et al. (2010) Plant Physiol. 153:611-621). Furthermore, mMDH anti-sense lines showed an increase in fruit desiccation (more H2O loss) and increased susceptibility to fungal infection. Centeno et al. (2011) Plant Cell 23:162-184. U.S. Patent Publication No. 20090123626 describes the use of MDH RNAi to reduce asparagine levels, which in turn lowers the level of acrylamide that accumulates upon processing-associated heating of the plant and plant products.
Thus, there remain needs for compositions and methods for altering expression of MDH genes, for example by targeted genomic modification of MDH genes, in plants for establishing stable, heritable genetic modifications in the plant and its progeny.